Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.     

Bmi1 is expressed in postnatal myogenic satellite cells, controls their maintenance and plays an essential role in repeated muscle regeneration

Satellite cells are the resident stem cell population of the adult mammalian skeletal muscle and they play a crucial role in its homeostasis and in its regenerative capacity after injury. We show here that the Polycomb group (PcG) gene Bmi1 is expressed in both the Pax7 positive (+)/Myf5 negative (-) stem cell population as well as the Pax7+/Myf5+ committed myogenic progenitor population. Depletion of Pax7+/Myf5- satellite cells with reciprocal increase in Pax7+/Myf5+ as well as MyoD positive (+) cells is seen in Bmi1-/- mice leading to reduced postnatal muscle fiber size and impaired regeneration upon injury. Bmi1-/- satellite cells have a reduced proliferative capacity and fail to re-enter the cell cycle when stimulated by high serum conditions in vitro, in keeping with a cell intrinsic defect. Thus, both the in vivo and in vitro results suggest that Bmi1 plays a crucial role in the maintenance of the stem cell pool in postnatal skeletal muscle and is essential for efficient muscle regeneration after injury especially after repeated muscle injury.     

Muscle satellite cells

Skeletal muscle satellite cells are quiescent mononucleated myogenic cells, located between the sarcolemma and basement membrane of terminally-differentiated muscle fibres. These are normally quiescent in adult muscle, but act as a reserve population of cells, able to proliferate in response to injury and give rise to regenerated muscle and to more satellite cells. The recent discovery of a number of markers expressed by satellite cells has provided evidence that satellite cells, which had long been presumed to be a homogeneous population of muscle stem cells, may not be equivalent. It is possible that a sub-population of satellite cells may be derived from a more primitive stem cell. Satellite cell-derived muscle precursor cells may be used to repair and regenerate damaged or myopathic skeletal muscle, or to act as vectors for gene therapy. CELL FACTS: (1) Number of cells in body: 2 x 10(7) to 3 x 10(7) myonuclei/g, 20-25 kg muscle in average man; 2 x 10(5) to 10 x 10(5) satellite cells/g, i.e. approximately 1 x 10(10) to 2 x 10(10) satellite cells per person. (2) Main functions: repair and maintenance of skeletal muscle. (3) Turnover rate: close to zero in non-traumatic conditions-high in disease or severe trauma.     

MyoD(-/-) satellite cells in single-fiber culture are differentiation defective and MRF4 deficient

MyoD-deficient mice are without obvious deleterious muscle phenotype during embryogenesis and fetal development, and adults in the laboratory have grossly normal skeletal muscle and life span. However, a previous study showed that in the context of muscle degeneration on a mdx (dystrophin null) genetic background, animals lacking MyoD have a greatly intensified disease phenotype leading to lethality not otherwise seen in mdx mice. Here we have examined MyoD(-/-) adult muscle fibers and their associated satellite cells in single myofiber cultures and describe major phenotypic differences found at the tissue, cellular, and molecular levels. The steady-state number of satellite cells on freshly isolated MyoD(-/-) fibers was elevated and abnormal branched fiber morphologies were observed, the latter suggesting chronic muscle regeneration in vivo. Single-cell RNA coexpression analyses were performed for c-met, m-cadherin, and the four myogenic regulatory factors (MRFs.) Most mutant satellite cells entered the cell cycle and upregulated expression of myf5, both characteristic early steps in satellite cell maturation. However, they later failed to normally upregulate MRF4, displayed a major deficit in m-cadherin expression, and showed a significant diminution in myogenin-positive status compared with wildtype. MyoD(-/-) satellite cells formed unusual aggregate structures, failed to fuse efficiently, and showed greater than 90% reduction in differentiation efficiency relative to wildtype. A further survey of RNAs encoding regulators of growth and differentiation, cell cycle progression, and cell signaling revealed similar or identical expression profiles for most genes as well as several noteworthy differences. Among these, GDF8 and Msx1 were identified as potentially important regulators of the quiescent state whose expression profile differs between mutant and wildtype. Considered together, these data suggest that activated MyoD(-/-) satellite cells assume a phenotype that resembles in some ways a developmentally "stalled" cell compared to wildtype. However, the MyoD(-/-) cells are not merely developmentally immature, as they also display novel molecular and cellular characteristics that differ from any observed in wild-type muscle precursor counterparts of any stage.     

Muscle satellite cell-specific genes identified by genetic profiling of MyoD-deficient myogenic cell

Satellite cells are committed myogenic progenitors that give rise to proliferating myoblasts during postnatal growth and repair of skeletal muscle. To identify genes expressed at different developmental stages in the satellite cell myogenic program, representational difference analysis of cDNAs was employed to identify more than 50 unique mRNAs expressed in wild-type myoblasts and MyoD-/- myogenic cells. Novel expression patterns for several genes, such as Pax7, Asb5, IgSF4, and Hoxc10, were identified that were expressed in both quiescent and activated satellite cells. Several previously uncharacterized genes that represent putative MyoD target genes were also identified, including Pw1, Dapk2, Sytl2, and NLRR1. Importantly, many genes such as IgSF4, Neuritin, and Klra18 that were expressed exclusively in MyoD-/- myoblasts were also expressed by satellite cells in undamaged muscle in vivo but were not expressed by primary myoblasts. These data are consistent with a biological role for activated satellite cells that induce Myf5 but not MyoD. Lastly, additional endothelial and hematopoietic markers were identified supporting a nonsomitic developmental origin of the satellite cell myogenic lineage.     

Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody

A novel monoclonal antibody, SM/C-2.6, specific for mouse muscle satellite cells was established. SM/C-2.6 detects mononucleated cells beneath the basal lamina of skeletal muscle, and the cells co-express M-cadherin. Single fiber analyses revealed that M-cadherin+ mononucleated cells attaching to muscle fibers are stained with SM/C-2.6. SM/C-2.6+ cells, which were freshly purified by FACS from mouse skeletal muscle, became MyoD+ in vitro in proliferating medium, and the cells differentiated into desmin+ and nuclear-MyoD+ myofibers in vitro when placed under differentiation conditions. When the sorted cells were injected into mdx mouse muscles, donor cells differentiated into muscle fibers. Flow cytometric analyses of SM/C-2.6+ cells showed that the quiescent satellite cells were c-kit-, Sca-1-, CD34+, and CD45-. More, SM/C-2.6+ cells were barely included in the side population but in the main population of cells in Hoechst dye efflux assay. These results suggest that SM/C-2.6 identifies and enriches quiescent satellite cells from adult mouse muscle, and that the antibody will be useful as a powerful tool for the characterization of cellular and molecular mechanisms of satellite cell activation and proliferation.     

Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment

Satellite cells are the primary stem cells in adult skeletal muscle, and are responsible for postnatal muscle growth, hypertrophy and regeneration. In mature muscle, most satellite cells are in a quiescent state, but they activate and begin proliferating in response to extrinsic signals. Following activation, a subset of satellite cell progeny returns to the quiescent state during the process of self-renewal. Here, we review recent studies of satellite cell biology and focus on the key transitions from the quiescent state to the state of proliferative activation and myogenic lineage progression and back to the quiescent state. The molecular mechanisms of these transitions are considered in the context of the biology of the satellite cell niche, changes with age, and interactions with established pathways of myogenic commitment and differentiation.     

A new look at the origin, function, and "stem-cell" status of muscle satellite cells

Muscle satellite cells have long been considered a distinct myogenic lineage responsible for postnatal growth, repair, and maintenance of skeletal muscle. Recent studies in mice, however, have revealed the potential for highly purified hematopoietic stem cells from bone marrow to participate in muscle regeneration. Perhaps more significantly, a population of putative stem cells isolated directly from skeletal muscle efficiently reconstitutes the hematopoietic compartment and participates in muscle regeneration following intravenous injection in mice. The plasticity of muscle stem cells has raised important questions regarding the relationship between the muscle-derived stem cells and the skeletal muscle satellite cells. Furthermore, the ability of hematopoietic cells to undergo myogenesis has prompted new investigations into the embryonic origin of satellite cells. Recent developmental studies suggest that a population of satellite cells is derived from progenitors in the embryonic vasculature. Taken together, these studies provide the first evidence that pluripotential stem cells are present within adult skeletal muscle. Tissue-specific stem cells, including satellite cells, may share a common embryonic origin and possess the capacity to activate diverse genetic programs in response to environmental stimuli. Manipulation of such tissue-specific stem cells may eventually revolutionize therapies for degenerative diseases, including muscular dystrophy.     

Rb1 gene inactivation expands satellite cell and postnatal myoblast pools

Satellite cells are well known as a postnatal skeletal muscle stem cell reservoir that under injury conditions participate in repair. However, mechanisms controlling satellite cell quiescence and activation are the topic of ongoing inquiry by many laboratories. In this study, we investigated whether loss of the cell cycle regulatory factor, pRb, is associated with the re-entry of quiescent satellite cells into replication and subsequent stem cell expansion. By ablation of Rb1 using a Pax7CreER,Rb1 conditional mouse line, satellite cell number was increased 5-fold over 6 months. Furthermore, myoblasts originating from satellite cells lacking Rb1 were also increased 3-fold over 6 months, while terminal differentiation was greatly diminished. Similarly, Pax7CreER,Rb1 mice exhibited muscle fiber hypotrophy in vivo under steady state conditions as well as a delay of muscle regeneration following cardiotoxin-mediated injury. These results suggest that cell cycle re-entry of quiescent satellite cells is accelerated by lack of Rb1, resulting in the expansion of both satellite cells and their progeny in adolescent muscle. Conversely, that sustained Rb1 loss in the satellite cell lineage causes a deficit of muscle fiber formation. However, we also show that pharmacological inhibition of protein phosphatase 1 activity, which will result in pRb inactivation accelerates satellite cell activation and/or expansion in a transient manner. Together, our results raise the possibility that reversible pRb inactivation in satellite cells and inhibition of protein phosphorylation may provide a new therapeutic tool for muscle atrophy by short term expansion of the muscle stem cells and myoblast pool.     

Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells

Notch signaling is a conserved cell fate regulator during development and postnatal tissue regeneration. Using skeletal muscle satellite cells as a model and through myogenic cell lineage-specific NICD(OE) (overexpression of constitutively activated Notch 1 intracellular domain), here we investigate how Notch signaling regulates the cell fate choice of muscle stem cells. We show that in addition to inhibiting MyoD and myogenic differentiation, NICD(OE) upregulates Pax7 and promotes the self-renewal of satellite cell-derived primary myoblasts in culture. Using MyoD(-/-) myoblasts, we further show that NICD(OE) upregulates Pax7 independently of MyoD inhibition. In striking contrast to previous observations, NICD(OE) also inhibits S-phase entry and Ki67 expression and thus reduces the proliferation of primary myoblasts. Overexpression of canonical Notch target genes mimics the inhibitory effects of NICD(OE) on MyoD and Ki67 but not the stimulatory effect on Pax7. Instead, NICD regulates Pax7 through interaction with RBP-Jκ, which binds to two consensus sites upstream of the Pax7 gene. Importantly, satellite cell-specific NICD(OE) results in impaired regeneration of skeletal muscles along with increased Pax7(+) mononuclear cells. Our results establish a role of Notch signaling in actively promoting the self-renewal of muscle stem cells through direct regulation of Pax7.     

Sphingomyelin levels in the plasma membrane correlate with the activation state of muscle satellite cells.

Satellite cells are responsible for postnatal growth, hypertrophy, and regeneration of skeletal muscle. They are normally quiescent, and must be activated to fulfill these functions, yet little is known of how this is regulated. As a first step in determining the role of lipids in this process, we examined the dynamics of sphingomyelin in the plasma membrane. Sphingomyelin contributes to caveolae/lipid rafts, which act to concentrate signaling molecules, and is also a precursor of several bioactive lipids. Proliferating or differentiated C2C12 muscle cells did not bind lysenin, a sphingomyelin-specific binding protein, but noncycling reserve cells did. Quiescent satellite cells also bound lysenin, revealing high levels of sphingomyelin in their plasma membranes. On activation, however, the levels of sphingomyelin drop, so that lysenin did not label proliferating satellite cells. Although most satellite cell progeny differentiate, others stop cycling, maintain Pax7, downregulate MyoD, and escape immediate differentiation. Importantly, many of these Pax7-positive/MyoD-negative cells also regained lysenin binding on their surface, showing that the levels of sphingomyelin had again increased. Our observations show that quiescent satellite cells are characterized by high levels of sphingomyelin in their plasma membranes and that lysenin provides a novel marker of myogenic quiescence.     

Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers

The satellite cell compartment provides skeletal muscle with a remarkable capacity for regeneration. Here, we have used isolated myofibers to investigate the activation and proliferative potential of satellite cells. We have previously shown that satellite cells are heterogeneous: the majority express Myf5 and M-cadherin protein, presumably reflecting commitment to myogenesis, while a minority is negative for both. Although MyoD is rarely detected in quiescent satellite cells, over 98% of satellite cells contain MyoD within 24 h of stimulation. Significantly, MyoD is only observed in cells that are already expressing Myf5. In contrast, a minority population does not activate by the criteria of Myf5 or MyoD expression. Following the synchronous activation of the myogenic regulatory factor+ve satellite cells, their daughter myoblasts proliferate with a doubling time of approximately 17 h, irrespective of the fiber type (type I, IIa, or IIb) from which they originate. Although fast myofibers have fewer associated satellite cells than slow, and accordingly produce fewer myoblasts, each myofiber phenotype is associated with a complement of satellite cells that has sufficient proliferative potential to fully regenerate the parent myofiber within 4 days. This time course is similar to that observed in vivo following acute injury and indicates that cells other than satellite cells are not required for complete myofiber regeneration.